Introduction

Klebsiella pneumoniae is crucial to the biotechnology industry, producing valuable chemicals such as 1,3-propanediol, 2,3-butanediol, 2-ketogluconic acid, and 3-hydroxypropionic acid. Along with other members of the Enterobacteriaceae family, K. pneumoniae is able to form a polysaccharides outer-capsule, which plays a critical role in protecting K. pneumoniae from phagocytic host cells (Schembri et al. 2004). The composition of the capsular polysaccharide is very much strain-dependent, and at least 78 serological types, based on capsule antigens, have been assigned to Klebsiella spp. (Pan et al. 2008).

In addition to these physiological functions, the capsule also hinders DNA penetration by electroporation, leading to low transformation efficiency and limiting genetic studies (Fournet-Fayard et al. 1995). In researching chemical production by K. pneumoniae, the capsular polysaccharide was found to increase broth viscosity, which impeded filtration, and added to financial burden of downstream processing (Guo et al. 2010).

Capsular polysaccharide synthesis occurs through complicated interactions involving enzymes encoded in gene clusters. To date, the genetic and biosynthetic pathways of K. pneumoniae capsule expression have yet to be fully characterized. The first K. pneumoniae cps gene cluster to be cloned was a serotype K2 strain, in which 19 open reading frames (ORF) were identified (Arakawa et al. 1995). Six genes located at the 5′ end (galF, acidPPc, wzi, wza, wzb, and wzc) and a single gene (gnd) located at the 3′ end were found to be highly conserved among K. pneumoniae species (Brisse et al. 2013; Rahn et al. 1999). The functions of a number of cps genes have been characterized in a serotype K1 strain (Ho et al. 2011).

Klebsiella pneumoniae CGMCC 1.6366 (TUAC01) was isolated for 1,3-propanediol production (Hao et al. 2008). An efficient gene replacement system in K. pneumoniae CGMCC 1.6366 has been exploited, and several mutants have been constructed (Wei et al. 2012). In the current study, the highly conserved wza gene of K. pneumoniae was deleted, creating a non-capsulated mutant, and the phenotype, transformation efficiency, chemical production of the non-capsulated mutant were investigated.

Materials and methods

Bacterial strains, plasmids, and primers

The bacterial strains and plasmids are listed in Table 1. The PCR primers are listed in Table 2.

Table 1 Bacterial strains and plasmids
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Table 2 Oligonucleotides used for PCR
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Determination of capsular serological type

The serological type was determined by wzi gene sequencing as per the method of Brisse et al. (2013). The wzi gene encodes an outer membrane protein involved in capsular attachment to the cell’s surface, and is highly conserved in K. pneumoniae species.

Construction of K. pneumoniae CGMCC 1.6366-derived mutants

Klebsiella pneumoniae and Escherichia coli were cultured in Luria–Bertani (LB) medium at 37 °C. When required, the medium was supplemented with ampicillin (50 μg mL−1), kanamycin (50 μg mL−1), apramycin (50 μg mL−1), or streptomycin (25 μg mL−1).

Klebsiella pneumoniae Δwza was constructed according to a previously described method (Wei et al. 2012). Briefly, the wza gene and flanking sequences of K. pneumoniae were amplified by PCR using the primer pair wza-s1/wza-a1. The PCR product was ligated with pMD18-T simple vector to generate pMD18-T-wza. Linear DNA with 39 and 40 nt homologous extensions flanking the apramycin resistance gene aac(3)IV were amplified from plasmid pIJ773 using the primer pair wza-FRT-s1/wza-FRT-a1. pMD18-T-Δwza was constructed by replacing the wza in plasmid pMD18-T-wza with the aac(3)IV cassette using the Red recombination system in E. coli. pMD18-T-Δwza was then used as a template for PCR preparation of linear DNA containing aac(3)IV with 500-bp homologous regions at either end. The linear DNA was transformed into K. pneumoniae/red, which hosts the plasmid pDK6-red. Homologous recombination between the linear DNA and the chromosome was facilitated by Red recombinase, and led to wza deletion in K. pneumoniae CGMCC 1.6366. The mutant was isolated on apramycin plates, and the primer pair Test773 and wza-s were used for PCR confirmation.

Klebsiella pneumoniae ΔbudAΔwza was constructed from K. pneumoniae Δwza. Chromosomal budA of K. pneumoniae Δwza was replaced with the aadA cassette, generating a budA and wza deletion strain. The aadA cassette was amplified from pMD18-T-ΔbudA by PCR using primers budA-s and budA-a.

Phenotype observation

Klebsiella pneumoniae Δwza and K. pneumoniae CGMCC 1.6366 were cultured in LB medium at 37 °C for 12 h. The culture broth was centrifuged 10,000×g for 10 min, and the supernatant discarded. The pellet was washed twice with deionized water and resuspended in the same volume of water. The cells were pelleted by centrifugation at 4000×g for 10 min, and the phenotypes were observed.

Transformation of K. pneumoniae by electroporation

The preparation of electrocompetent cells and the conditions used for electroporation were as previously described (Wei et al. 2012). Ethylenediaminetetraacetic acid (EDTA)-treated electrocompetent cells were prepared by adding 0.7 mM EDTA to the cell culture when the OD600 reached 0.2. Plasmid pDK6 was used to transform cells for transformation efficiency determination. All experiments were conducted in triplicate.

Medium and culture conditions

The fermentation medium and culture conditions used for 1,3-propanediol production were as previously described (Hao et al. 2008).

The composition of the fermentation medium used for 2,3-butanediol production is as follows: 50 g glucose L−1, 4 g corn steep liquor L−1, 5 g (NH4)2SO4 L−1, 3 g sodium acetate L−1, 0.4 g KCl L−1, and 0.1 g MgSO4 L−1. For the seed culture, 250 mL flasks containing 50 mL LB broth were incubated in a rotary shaker at 37 °C and 200 rpm overnight. The 50 mL of seed culture was inoculated into a 5 L bioreactor (BIOSTAT-A plus Sartorius) with a working volume of 3 L. The air supplement, agitation, and culture temperatures were 4 L min−1, 250 rpm, and 37 °C, respectively. The culture pH was maintained at pH 6 by the automated addition of 10 M NaOH. 600 g glucose L−1 solution was fed into the bioreactor when the glucose level in the medium decreased to 20 g L−1.

The composition of the fermentation medium used in 2-ketogluconic acid production was as described previously (Wei et al. 2013). A two-stage fermentation strategy was used in 2-ketogluconic acid production. During the first stage of fermentation (4 h), the culture was maintained at pH 7.0, and agitated at 500 rpm. Upon completion of the first fermentation stage, the pH was adjusted to pH 5.0, and the bioreactor was agitated at 800 rpm. NH3 solution was used to maintain pH.

All fermentation experiments were conducted in triplicate.

Analysis of biomass, substrate, and metabolic products

Biomass concentration was determined at 600 nm using UV-visible spectroscopy system (Beckman DU730, USA). Glucose, glycerol, 1,3-propanediol, 2,3-butanediol, and 2-ketogluconic acid concentrations were measured by HPLC as previously described (Hao et al. 2008; Wei et al. 2013).

Fermentation broth viscosity measurements

Fermentation broth viscosity was measured with a Brookfield viscometer at 30 °C and 100 rpm. The viscosity of the supernatant was also measured. Briefly, the fermentation broth was centrifuged at 10,000 × g for 10 min, the supernatant recovered, and the viscosity of the supernatant determined as for fermentation broth.

Results

Gene type and serological type of the capsule

The cps gene cluster of K. pneumoniae CGMCC 1.6366 (GenBank accession no: KJ128966) is 24862 bp and contains 20 ORFs (Fig. 1). In addition to the seven highly conserved genes (galF, acidPPc, wzi, wza, wzb, wzc, and gnd) of K. pneumoniae, six genes (wcaF, gmd, wcaG, wcaH, wcaI, and wcaJ) were found to have high homology with alleles of K. pneumoniae NTUH K-2044. The remaining seven genes (orf7, orf8, orf9, orf10, orf11, orf12, and orf19) did not exhibit high homology with any sequence in GenBank. The functions of these genes were predicted from the protein sequence alignment in GenBank as follows: orf7, polysaccharide biosynthesis protein; orf8, a putative pyruvyltransferase; orf9, a putative O-antigen polymerase; orf10, orf11, orf12, three glycosyl transferase-GTB-type superfamily proteins; and orf19, a putative acetylase.

Fig. 1
figure 1

The cps gene cluster of K. pneumoniae CGMCC 1.6366. Genes highly conserved (blue), genes have alleles (red), remaining genes (black)

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The capsular serological type of K. pneumoniae CGMCC 1.6366 was determined using wzi gene sequencing. The alignment of the wzi gene of K. pneumoniae CGMCC 1.6366 with 135 alleles of Klebsiella spp. presented by Brisse et al. (2013) demonstrated that K. pneumoniae CGMCC 1.6366 has a unique wzi sequence, which clusters closely with alleles of K. oxytoca strain wzi26-K27 and K. terrigena strain wzi68. wzi26-K27 strain was of serological type K27, while no serological type was assigned to strain wzi68.

Phenotype of K. pneumoniae Δwza

Klebsiella pneumoniae Δwza and K. pneumoniae ΔwzaΔbudA were constructed as described in the section of Materials and methods. A greater volume of water is retained when the capsule is attached to the cell surface, affecting cell density. Cell suspensions of K. pneumoniae Δwza and K. pneumoniae CGMCC 1.6366 post-centrifugation are presented in Fig. 2.

Fig. 2
figure 2

Cell suspensions of K. pneumoniae CGMCC 1.6366 (A) and K. pneumoniae Δwza (B) post-centrifugation

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Figure 2 clearly illustrates that the majority of cells remained in suspension for K. pneumoniae CGMCC 1.6366. In contrast, the majority of cells for K. pneumoniae Δwza had settled to the bottom of the centrifuge tube. These results indicate that capsule formation was significantly diminished in K. pneumoniae Δwza.

Capsule effect on the efficiency of transformation by electroporation

Klebsiella pneumoniae Δwza, K. pneumoniae CGMCC 1.6366, and EDTA-treated K. pneumoniae CGMCC 1.6366 electrocompetent cells were transformed with pDK6. The transformation efficiency was determined, and the results shown in Fig. 3.

Fig. 3
figure 3

Transformation efficiency of K. pneumoniae Δwza, K. pneumoniae CGMCC 1.6366 (wild strain), and EDTA-treated K. pneumoniae CGMCC 1.6366. The transformation experiments were done in triple and the error bars represent the standard deviation

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Transformation efficiency of pDK6 into K. pneumoniae CGMCC 1.6366 was 5.5 × 104 CFU μg−1 DNA. The addition of EDTA to electrocompetent cells increased the transformation efficiency to 2.6 × 105 CFU μg−1 DNA, while the transformation efficiency of the wza mutant was 6.4 × 105 CFU μg−1 DNA. Deletion of the wza gene resulted in diminished/annihilated capsule formation, which removed the barrier to DNA penetration, and consequently improved transformation efficiency.

Capsule effect on 1,3-propanediol production

Klebsiella pneumoniae CGMCC 1.6366 and K. pneumoniae Δwza were cultured under micro-aerobic conditions, with glycerol as a carbon source, for 1,3-propanediol production, in which the formation of 1,3-propanediol and cell growth were measured at regular intervals for 34 h (Fig. 4).

Fig. 4
figure 4

1,3-Propanediol production by K. pneumoniae CGMCC 1.6366 and K. pneumoniae Δwza. a: 1,3-propanediol formation; b: Cell growth. K. pneumoniae CGMCC 1.6366 (open circle); K. pneumoniae Δwza (filled circle). The fermentations were done in triple and the error bars represent the standard deviation

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Klebsiella pneumoniae CGMCC 1.6366 produced a maximum of 52.2 g 1,3-propanediol L−1 at 34 h, while K. pneumoniae Δwza produced only 47.9 g L−1. Similar growth curves were observed between the two strains with K. pneumoniae CGMCC 1.6366 achieved its greatest cell dry weight (CDW) 3.69 g L−1 at 24 h, and K. pneumoniae Δwza CDW 3.6 g L−1 at 21 h.

Capsule effect on 2,3-butanediol production

Klebsiella pneumoniae CGMCC 1.6366 and K. pneumoniae Δwza were cultured under micro-aerobic conditions, with glucose as a carbon source, for 2,3-butanediol production. The results are presented in Fig. 5.

Fig. 5
figure 5

2,3-Butanediol production by K. pneumoniae CGMCC 1.6366 and K. pneumoniae Δwza. A: 2,3-butanediol formation; B: Cell growth. K. pneumoniae CGMCC 1.6366 (open circle); K. pneumoniae Δwza (filled circle). The fermentations were done in triple and the error bars represent the standard deviation

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Klebsiella pneumoniae CGMCC 1.6366 produced a maximum of 51.2 g 2,3-butanediol L−1 at 34 h; K. pneumoniae Δwza produced 30.7 g L−1. Throughout the duration of this experiment, cell growth for K. pneumoniae Δwza was less than the wild strain. The greatest cell dry weights were 3.24 and 4.65 g L−1 for K. pneumoniae Δwza and K. pneumoniae CGMCC 1.6366, respectively.

Capsule effect on 2-ketogluconic acid production

Klebsiella pneumoniae ΔbudA can’t synthesize 2,3-butanediol, but 2-ketogluconic acid accumulates to a high level. Therefore, a budA and wza double gene deletion strain K. pneumoniae ΔbudAΔwza was constructed and used to evaluate 2-ketogluconic acid productivity. The two strains were cultured under aerobic conditions, with glucose as a carbon source, for 2-ketogluconic acid production. The fermentations proceeded through a two-stage strategy and the results are shown in Fig. 6.

Fig. 6
figure 6

2-Ketogluconic acid production by K. pneumoniae ΔbudA and K. pneumoniae ΔbudAΔwza. a 2-Ketogluconic acid formation; b Cell growth. K. pneumoniae ΔbudA (open circle); K. pneumoniae ΔbudAΔwza (filled circle). The fermentations were done in triple and the error bars represent the standard deviation

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The total amounts of 2-ketogluconic acid produced by K. pneumoniae ΔbudA and K. pneumoniae ΔbudAΔwza at 30 h culture were 175.9 and 158.5 g L−1, respectively. Cell growth for K. pneumoniae ΔbudAΔwza was slower compared with K. pneumoniae ΔbudA.

Fermentation broth viscosities

The viscosities of 1,3-propanediol, 2,3-butanediol, and 2-ketogluconic acid fermentation broths were measured on continuous fermentation (Table 3).

Table 3 Viscosities of 1,3-propanediol, 2,3-butanediol, and 2-ketogluconic acid fermentation broths
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The viscosities of fermentation broths are dependent on solute concentration; the higher the concentration of capsular polysaccharide in the broth, the greater the viscosity. As shown in Table 3, the viscosities of the fermentation broths and supernatants of K. pneumoniae CGMCC 1.6366 or K. pneumoniae ΔbudA were consistently high. These results suggested that in addition to the cell surface, some of the capsular polysaccharide produced was dissolved in the fermentation broth. A reduction in capsular polysaccharide formation for the wza mutants (K. pneumoniae Δwza and K. pneumoniae ΔbudAΔwza) lowered the viscosities of fermentation broths; the viscosities were 150% that of water [water viscosity is 0.80 cP at 30 °C (Dean 1999)].

Discussion

Klebsiella pneumoniae is an important industrial bacterium, used in the production of many chemicals. Unfortunately, public perception associates K. pneumoniae with pneumonia, and its use, therefore, prompts safety concerns from the public. The pathogenic mechanism of this infectious disease is yet to be deciphered (Struve et al. 2005). The capsule has been identified as a virulence factor of K. pneumoniae, and capsular serotypes K1 and K2 are considered the predominant virulent strains (Chuang et al. 2006). Meanwhile, the capsule is not equivalent to pathogenicity. K. pneumoniae 342 is a nitrogen-fixing endophyte and has capsule, and mouse models have proved that the pathogenicity was attenuated in the strain (Fouts et al. 2008). Seven genes and a section of non-coding sequence in the cps gene cluster of K. pneumoniae CGMCC 1.6366 don’t share high sequence homology with nucleotide sequences of any allele in GenBank. Collectively, these sequences comprise 37.5% of the total length of the cps gene cluster. Serological typing indicated that K. pneumoniae CGMCC 1.6366 closely aligned with wzi26-K27 and wzi68 strains; non-K. pneumoniae species of genus Klebsiella. These results indicated critical divergences between K. pneumoniae CGMCC 1.6366 and pathogenic strains.

A highly conserved block of genes, wzi-wza-wzb-wzc, have been identified in the cps gene cluster of E. coli and Klebsiella spp. wza encodes an outer membrane protein, wzb encodes a cytoplasmic phosphatase and wzc encodes an ATP-binding protein. These proteins represent a common translocation-surface assembly pathway for cell surface polysaccharides (Rahn et al. 1999). Since wza is a highly conserved gene in cps gene clusters, it was targeted for deletion, and a non-capsulated mutant pneumoniae Δwza was generated. The rmpA2 gene encodes an activator for capsular polysaccharide synthesis. This gene was deleted to produce a non-capsulated variant of K. pneumoniae CG43S3, which sedimented much faster than its parental strain (Lai et al. 2003). This result is in agreement with the current investigation on K. pneumoniae Δwza. K. pneumoniae J2B strain has reduced lipopolysaccharide formation (Arasu et al. 2011), and has similar sedimentation properties to K. pneumoniae Δwza constructed in this study.

A wza deletion mutant of K. pneumoniae NTUH K2044 underwent mucoviscosity loss and a significant decrease in virulence (Ho et al. 2011), which was also observed in K. pneumoniae Δwza. The uge gene encodes UDP galacturonate 4-epimerase, and the mutations in the uge gene affect polysaccharide and lipopolysaccharide processing, resulting in capsule loss. uge mutants have been shown to be completely avirulent in two different animal models (Regue et al. 2004). Therefore, K. pneumoniae Δwza appears to be a safer strain for use in industrial applications.

The transfer of exogenous DNA into cells is essential for genetic and molecular biology studies, and electroporation is a common method practiced in many laboratories worldwide. The electroporation method was developed using E. coli K12. However, transformation rate using this method for wild strains is quite low. Transformation of K. pneumoniae 21 with pBR328 using electroporation resulted in 104 transformants μg−1 DNA (Fournet-Fayard et al. 1995). The transformation rate for K. pneumoniae CGMCC 1.6366 with pDK6 obtained in the current study was of the same order of magnitude. The exact mechanism for DNA uptaken by electroporation is not entirely clear, but plasmid DNA must breach the bacterial membrane. The capsule acts as a barrier to plasmid DNA penetration by electroporation. The addition of chemicals such as EDTA, which reduced capsule formation, enhanced transformation efficiency to 108 transformants μg−1 DNA in K. pneumoniae 21 (Fournet-Fayard et al. 1995). For K. pneumoniae CGMCC 1.6366, the transformation efficiency increased to 2.64 × 105 CFU μg−1 DNA with the inclusion of EDTA. The wza mutant had the highest transformation efficiency.

1,3-Propanediol, 2,3-butanediol, and 2-ketogluconic acid are all valuable chemicals produced by K. pneumoniae. An approximate 10% decrease in 1,3-propanediol and 2-ketogluconic acid productivity was observed for the non-capsulated mutant when compared with wza wild strains. The rates of cell growth during 1,3-propanediol and 2-ketogluconic acid production were similar for mutant and wza wild strains. This is in agreement with studies conducted on cps mutants of K. pneumoniae NTUH K2044, whereby the authors proposed that the capsule was important for pathogenicity but not for growth (Ho et al. 2011).

Unfortunately, 2,3-butanediol productivity of K. pneumoniae Δwza was markedly lower than that of the wild strain. There is no overlap between the 2,3-butanediol synthesis pathway and the cps cluster, hence the mechanism for the decrease in 2,3-butanediol productivity observed for the wza mutant is difficult to deduce. However, it was shown in other research that the yield of 2,3-butanediol, which was 52.4 g L−1, with the non-capsulated strain was close to the yield of this study. (Rathnasingh et al. 2012).

In the downstream processing of 1,3-propanediol and 2,3-butanediol fermentation broths, cells, and other solid impurities must be removed from the broth to obtain a clear liquid for further purification processes (Hao et al. 2006; Xiu and Zeng 2008), and same applies to 2-ketogluconic acid broth. Filtration techniques, especially membrane filtration, are commonly used for clarifying fermentation broth. The viscosity of the broth is a key parameter in the filtration process. Viscosity has a positive relationship with energy consumption, and a negative relationship with membrane flux (Van and Zydney 2001). The capsule polysaccharide produced in the broth greatly contributes to viscosity and impedes filtration. Fermentation broths produced by K. pneumoniae Δwza and K. pneumoniae ΔbudAΔwza had low viscosities; a desirable quality for filtration.